Recombinant DNA methods permit the construction of nucleic acid eukaryotic expression cassettes encoding a product of interest. These expression cassettes are then introduced into the cytoplasm of eukaryotic cells using methods known in the art. However, a major difficulty in the expression of these expression cassettes is that the nucleic acid encoding the product of interest must be exported into the nucleus where the eukaryotic transcription machinery resides. Those expression cassettes that remain in the cytoplasm are not transcribed due to the lack of a cytoplasmic RNA polymerase that can transcribe the cassette.
One strategy to increase levels of expression of the product of interest from expression cassettes following non-viral cell transfection involves the use of a cytoplasmic expression system (Gao and Huang (1993) Nucleic Acids Res. 21: 2867-2872). The advantage of such a system is that it bypasses the need for nuclear delivery of plasmid DNA, a major obstacle in present day expression systems and in gene therapy. The efficiency of nuclear delivery following intracellular delivery is very low and is dependent on the size of the plasmid DNA molecule (Hagstrom et al. (1997) J Cell Sci. 110: 2323-2331). The addition of nuclear localization signals to plasmid DNA, has been shown to enhance transfection, but with limited success (Arohsohn and Hughes (1998) J. Drug Targeting 5: 163-169). The primary barrier to nuclear delivery of plasmid DNA is thought to be the nuclear membrane as plasmid DNA enters the nucleus more efficiently in mitotic or dividing cells, during the breakdown of the nuclear envelope (Coonrod et al. (1997) Gene Ther. 4: 1313-1321). As a result, gene expression following transfection is much higher in dividing than non-dividing cells (Vitadelo et al. (1994) Hum. Gen. Ther. 5: 11-18; Miller et al., (1992) Mol. Cell. Biol. 10: 4239-4242). A further limitation of nuclear expression systems is the finite, saturable limit to the amount of DNA that can be taken up by the nucleus under any condition (Brisson et al. (1999) Human Gene Therapy 10: 2601-2613).
Attempts have been made to incorporate non-host RNA polymerase promoters and genes encoding RNA polymerases with expression systems to overcome the above limitations. More particularly, these limitations have led to the development of strategies that do not require nuclear localization of DNA. One of these involves the use of bacteriophage T7 RNA polymerase (T7 RNAP). T7 RNAP is a single polypeptide enzyme that mediates transcription in the cytoplasm with high promoter specificity and efficiency (Davanloo et al. (1984) Proc. Natl. Acad. Sci., U.S.A. 81: 2035-2039). These properties have facilitated the development of a T7 based cytoplasmic expression system. Such systems require cytoplasmic delivery of both a plasmid construct containing a gene of interest under transcriptional control of the T7 promoter and a source of the T7 polymerase. Initial studies involved co-transfection of cells with plasmids carrying T7 controlled genes and purified T7 RNAP protein. These systems were able to bypass the need for the nuclear transcription machinery and yielded high levels of gene expression (Gao and Huang (1993)). Due to the instability of the T7 RNAP protein, however, the resulting gene expression was short lived, and considerable T7 RNAP associated cytotoxicity was observed (Gao and Huang (1993)).
These studies led to the development of the T7 polymerase autogene. This system consists of a T7 RNAP gene driven by its own T7 promoter, along with a reporter gene, on different plasmids. When cells were co-transfected with these constructs and purified T7 RNAP protein, rapid and sustained levels of reporter protein were detected. The T7 autogene was able to replenish its supply of T7 RNAP, resulting in sustained gene expression (Chen et al. (1994) Nucleic Acids Res. 22: 2114-2120). While these autogenes are effective, the transfection cocktail is difficult to prepare and, in practice, has been shown to be cytotoxic. To overcome these problems, a dual promoter autogene was created (Brisson et al. (1999) Gene Ther. 6: 263-270). This construct contained a T7 RNAP gene in control of both T7 (cytoplasmic) and CMV (nuclear) promoters. This construct when taken up into the nucleus resulted in low levels of T7 RNAP being produced. The T7 RNAP produced in the nucleus in turn is able to transcribe the cytoplasmic plasmid, which is the major portion of plasmid in the cell. This in turn leads to more T7 RNAP being produced which acts to amplify the production of more T7 RNAP and the reporter gene product. Theoretically, one plasmid incorporated into the nucleus would be sufficient to activate and induce high levels of gene expression from thousands of cytoplasmic plasmids. However, this effect is limited to the cell in which the RNAP is being expressed. Other cells in which DNA is not being expressed in the nucleus, do not show the autogene effect.
Thus, a need exists in the art for nucleic acids, nucleic acid compositions, and methods that permit a RNAP to enter a cell containing cytoplasmic expression cassettes and to express the nucleic acid in the cassette that is under the control of a RNA polymerase promoter. The present invention fulfills these and other needs in the art.